The development of safe and effective vaccines is essential to prevent systemic infections, which continue to cause high rates of morbidity and mortality throughout the world. To prevent and ultimately eradicate a wide variety of systemic infections caused by bacterial pathogens or microorganisms with neutral polysaccharides, universal immunization of infants and adults with suitable vaccines, which are capable of eliciting safe, effective, and long-lasting immunity, is needed. Because the incidence of diseases caused by invasive bacterial pathogens is highest in children under two years of age, effective immunization must begin early and optimally be completed within the first half year of life. Subsequent booster immunizations at about fifteen months and five years may also be advocated.
Surface polysaccharides serve as essential virulence factors and protective antigens for invasive bacteria which infect the bloodstream as their primary pathogenic event (39, 41). These surface antigens include capsular polysaccharides of both Gram-negative and Gram-positive bacteria and the lipopolysaccharides of Gram-negative bacteria. Although most of these purified polysaccharides elicit protective levels of antibodies in healthy adults, both capsular polysaccharides and lipopolysaccharides have immunologic properties or pharmacologic activities that limit their use as vaccines. Capsular polysaccharides are known in the medical literature and are polymers of sugars, sugar acids, amino sugars, polyhydric alcohols, and sugar phosphates. The capsular polymers may contain linkages other than glycosidic linkages and constituents other than the abovementioned sugars.
The immunologic properties of capsular polysaccharides which limit their use in infants and children, the age group with the highest attack rate of systemic infections caused by capsular pathogens, is their age-related immunogenicity (i.e. the ability to elicit the production of antibodies) and their T cell independence (i.e. lack of a statistically significant rise in antibody levels following re-injection or booster response). Pn14 has the abovementioned properties which exclude its usefulness as a vaccine for infants and children. That is, by itself, Pn14 does not elicit protective levels of antibodies in children up to four years of age (age-related immunogenicity) and does not elicit a booster response following m-injection (T cell independence) (40, 56, 57). In healthy adults, injection of Pn14 elicits protective levels of antibodies; however, re-injection of Pn14 does not elicit a booster response (40, 57).
To provide protection against some capsulated bacterial pathogens of infants and children, established vaccines have been used which comprise proteins such as diphtheria or tetanus toxoids or other potentially protective antigens such as the toxoid of Pseudomonas aeruginosa exotoxin A or pneumococcus hemolysin attached to polysaccharides (1, 6, 10, 12, 15, 22, 33, 41, 49). It has been estimated that induction of protective levels of antibodies to approximately ten bacterial polysaccharides would potentially result in a substantial decrease in serious infections of infants (2-4). However, complex formulation of conjugates containing the approximately six to ten polysaccharides of medical interest would require more carder proteins than are currently available as vaccines, for example, diphtheria toxoid or tetanus toxoid. There is a limit to the number of saccharides that can be bound to a single protein because of the possibility of immune suppression due to carrier protein overload of the immune system. Others have worked with conjugates for prevention of systemic infections caused by medically important organisms such as Haemophilus infiuenzae type b or various serotypes of Streptococcus pneumoniae (6, 10, 15, 33, 34, 41, 49, 50) in an effort to afford immune protection in mammals.
Streptococcus pneumoniae (pneumococci) are a major cause of otitis media, pneumonia, meningitis, and other invasive diseases which cause mortality and morbidity throughout the world (2-4, 7, 40, 47). Otitis media is the most common disease caused by this pathogen in infants and children (2-4, 26, 40). Several large surveys have reported that of the 87 known capsular polysaccharide types (i.e. serotypes) of Streptococcus pneumoniae, Pn14 is the one type which is isolated most frequently from patients of all ages (2-4, 40). The capsular polysaccharide of Streptococcus pneumoniae type 14 is also called pneumococcus type 14 and is composed of a neutral (i.e. a non-charged or non-negatively charged), repeating tetrasaccharide (24): ##STR1##
Pneumococcus type 14 is a comparatively poor immunogen among the pneumococcus capsular polysaccharides. Pn14 is a less-than-optimal immunogen in the 23-valent vaccine for adults (2, 7, 40, 54, 55). Also, Pn14 does not elicit protective levels of antibodies in human infants and young children--the age groups with the highest attack rate of diseases caused by Streptococcus pneumoniae (1, 2, 15, 40, 56, 57). In addition, except for humans, Streptococcus pneumoniae type 14 is of limited virulence and is not pathogenic for animals, including mice. Pn14 elicits serum antibodies in humans only (25, 27).
Bordetella pertussis, the microorganism that causes whooping cough or pertussis, is a highly contagious upper respiratory pathogen (36). Bordetella pertussis produces a protein toxin called pertussis toxin (PT) which elicits a variety of severe physiological and cellular responses including induction of lymphocytosis, sensitization to the lethal effects of histamine, and stimulation of insulin secretion, to name a few (28-31, 36, 42, 43, 45). The physiologic and cellular actions of PT appear to be mediated by interference with cyclic nucleotides (45). Laboratory and clinical data have shown that pertussis toxin is both a virulence factor (36) and a protective antigen (10, 31, 33, 43, 47). An inactivated toxin (i.e. a toxoid) is an essential component of acellular vaccines for pertussis (17, 18, 31, 32, 36, 46). Both laboratory and clinical data have shown that serum antibodies confer protective immunity to pertussis (29, 30, 32, 42) and have provided evidence that pertussis toxin is essential for vaccine-induced protective immunity to pertussis.
Although PT can elicit the production of protective antibodies, PT has several properties which make it difficult to use as a carder protein. For example, PT is mostly insoluble at pH values between about 4 and 8, which are optimal for carrying out many conventional coupling reaction procedures. In addition, PT is composed of six polypeptides that are not covalently bound.
The synthesis of saccharide-protein conjugate vaccines has been reported since 1929 (5, 23). The experimental data from these reports established the principle that the immunogenicity (i.e. the ability to elicit serum antibodies) of a saccharide may be increased following its covalent attachment to a protein. However, these initial synthetic schemes used chemicals and routes of immunization in animals that are neither appropriate for use nor suitable for clinical investigation in humans. The synthesis of a clinically-acceptable saccharide-protein conjugate vaccine was first reported in 1980 (53). The 1980 report described the covalent binding of saccharide to protein by first reacting the linking molecule, adipic acid dihydrazide (ADH), with the protein. Next, this derivatized protein was bound to the polysaccharide. In a later scheme using Haemophilus infiuenzae type b and pneumococcal 6A polysaccharide, the polysaccharide was first derivatized with ADH and then was covalently bound to the protein (10).
Since 1980, several other synthetic schemes for preparing saccharide-protein conjugates have been reported (reviewed in 41). ADH has been used as a linking molecule for a number of saccharides other than Pn14, which have properties different from those of Pn14 (11, 12, 13, 22, 34, 52).
Conjugates of Pn14 have been reported using reductive amination to couple Pn14 to diptheria toxoid (1) or using carbodiimide-mediated coupling at pH greater than 4, followed by overnight dialysis at neutral pH (49, 50).
The production of a saccharide conjugate with pertussis toxin was reported as a presentation at a conference (38). The saccharide group, group C capsular polysaccharide of Neisseria meningitidis (meningococcus), was an oligosaccharide prepared by acid pyrolysis. The resultant saccharide was derivatized with the N-hydroxysuccinimide ester of adipic acid. The derivative was dissolved in dimethyl sulfoxide and was added to pertussis toxin which was also dissolved in dimethyl sulfoxide. The reaction mixture (i.e. the derivatized oligosaccharide and pertussis toxin) was then passed through a Sephadex G-100 column. Polyacrylamide gel electrophoresis (PAGE) of the reaction mixture showed two major components which differed from the profiles of the group C meningococcal oligsaccharide and from that of PT. A critical reading of this conference report shows that the sections purporting to show data and results provided no experimental proof that the saccharide was covalently bound (i.e. chemically bonded) to the PT. Thus, there is a lack of experimental evidence that chemical bonding of the saccharide to the PT occurred. Further, although it is well known that dimethyl sulfoxide is an organic solvent which has the property of denaturing proteins, the effect of dimethyl sulfoxide on the migration of PT in PAGE was neither mentioned nor identified. Gel chromatography was used to compare the alleged conjugate with PT alone. Using this technique, there was no difference in the chromatographic profiles of the alleged conjugate and the PT. Moreover, an unconventional technique described in the report as "affinity" electrophoresis was used to show that PT was chemically combined with the group C meningococcal oligosaccharide. However, the results of the "affinity" electrophoresis were neither shown by illustration nor described in the text. Further, the report provided no mention of the use of controls, such as other structurally similar polysaccharides or linear homopolymers that are negatively-charged. Lastly, the report did not consider or mention injecting the "conjugate" into animals or humans to verify that 1) a serum antibody response was elicited by the "conjugate"; 2) serum antibodies were generated against both the group C meningococcal oligsaccharide and the PT; or 3) that serum antibody response to the saccharide had improved or changed. Both unsupported and uncontrolled data, and conclusions drawn without proper experimental findings, lead to the determination that no valid scientific evidence was presented to show that the group C meningococcal saccharide was bound to PT, or that the reported preparation had enhanced the immunogenicity of the saccharide.
U.S. Pat. No. 5,085,862, issued Feb. 4, 1992, discloses the production of defined mutant holotoxins of pertussis toxin using site directed mutagenesis. The toxin analogues as described are immunoprotective in mice. U.S. Pat. No. 4,788,058, issued Nov. 29, 1988, describes a method of preparing an immunogenic preparation of pertussis toxin treated in the form of an intimate admixture with filamentous hemagglutinins. U.S. Pat. No. 4,673,574, issued Jun. 16, 1987, describes an immunogenic conjugate which is the reductive amination product of a capsular polymer fragment having a reducing end and derived from a bacterial capsular polymer of a bacterial pathogen. The problems associated with the use of PT, such as its insolubility under conditions of neutral pH, are neither addressed nor surmounted.